I. Field of the Invention
This invention relates to a method of assaying a target substance.
II. Description of the Related Art
Various methods for assaying a target substance such as a biological substance have been developed. Among these, the most sensitive methods are those utilizing the specific binding reaction between a target substance and a substance which specifically reacts with the target substance (hereinafter referred to as "specifically binding substance" for short), for example, specific binding reaction between an antigen and an antibody, a sugar chain and lectin, biotin and avidin, Protein A and IgG, a hormone and a receptor, or an enzyme and a substrate.
In general, the specific binding substance labelled with a marker (hereinafter referred to as "labelled substance" for short) is utilized, and the signal from the labelled substance, which varies depending on the amount of the target substance to be assayed, is measured so as to assay the target substance.
In particular, in a conventional method, a target substance directly or indirectly fixed to a support is reacted with the labelled substance to fix the complex therebetween on the support, and the signal from the labelled substance on the support, which varies depending on the amount of the target substance to be assayed, is measured so as to assay the target substance.
Examples of this type of methods include an assay in which a protein component (target substance) separated by electrophoresis is transferred to a nitrocellulose membrane from the electrophoresis gel, and a labelled substance (e.g., a labelled antibody) is then reacted with the protein on the nitrocellulose membrane, followed by measuring the signal from the labelled substance; an assay in which a target substance on a thin layer chromatography (TLC) plate, such as a lipid fractionated by TLC is reacted with a labelled substance and the signal from the labelled substance is measured; an assay in which a DNA and a labelled DNA complementary to the DNA are reacted to detect a signal from the labelled substance; and immunohistochemical staining methods.
By these assays, not only the quantification of the target substance or the reactivity between the target substance and the specifically binding substance, but also much information about the property and/or the state of existence of the target substance and/or the specifically binding substance may be obtained. For example, by the assay in which a biological target substance such as a protein or a nucleic acid transferred to a nitrocellulose membrane from the gel after electrophoresis, or a lipid fractionated on a TLC plate is reacted with a labelled substance and the signal from the labelled substance is measured, the location of the target substance may be determined, and the molecular weight, isoelectric point and/or the polarity of the target substance may be determined from the mobility of the target substance.
By the immunohistochemical staining method, information such as the location and/or the state of existence of the target substance in a tissue may be obtained.
In the above-mentioned assay in which the target substance directly or indirectly fixed on a support is assayed by measuring the signal from the complex between the target substance and the labelled substance, since the amount of the target substance is very small, it is required that the marker be detected with high sensitivity, and in order to obtain more information about the target substance, it is required that the marker be detected with high resolution.
To satisfy these requirements, radioactive isotopes, fluorescent substances, luminescent substances and enzymes are conventionally utilized as the marker.
When a radioactive substance is utilized, however, the radioactivity of the marker is decreased with time, discarding the radioactive marker is troublesome and there is a danger for an operator to be exposed to the radioactivity. Further, the equipment is expensive. Still further, when the signal from the marker fixed on the support is measured, time consuming and troublesome operations such as exposure of photographic material and development thereof are needed.
Similarly, when a fluorescent or luminescent marker is employed, a special equipment is required.
On the other hand, in cases where an enzyme is employed as a marker, the operation is relatively simple, the generated color can easily be visualized and the quantification of the color can easily be attained. As the labelling enzyme, peroxidase, alkaline phosphatase and .beta.-galactosidase are conventionally employed. In the assay in which a pigment is formed by an enzyme reaction on the support on which the complex is fixed, peroxidase is mainly used as the labelling enzyme and diaminobenzidine, o-dianizidine or 4-chloro-1-naphthol is conventionally used as the substrate.
Diaminobenzidine and o-dianizidine, however, have a drawback in that they have strong toxicity and they give high background. 4-chloro-1-naphthol has unsatisfactory sensitivity in view of obtaining more information about the target substance, although it gives higher sensitivity than the other two substrates.
The present inventors have developed a method of assaying a target substance with high sensitivity and high resolution, which can be carried out quickly, and the method is disclosed in Japanese Patent Disclosure (Kokai) No. 150723/86. In this method, a complex between a target substance to be assayed and a labelled substance labelled with peroxidase is fixed on a support, a pigment is formed by the enzyme reaction of the peroxidase and the pigment is deposited on the substrate, wherein hydrogen peroxide, an aromatic primary amine and a phenolic compound are used as the substrate.
The support employed in this type of method is usually in the form of a membrane and nitrocellulose membrane which is hydrophilic is most widely used as the support.
However, in the above-described sensitive method wherein hydrogen peroxide, an aromatic primary amine and a phenolic compound are used as the substrates, if a nitrocellulose membrane is used as the support, the pigment formed and deposited on the nitrocellulose membane is decomposed with time, especially by the action of light. The decomposition of the pigment is a serious problem on the accuracy of the assay and on the long time storage of the record.